1. Field of the Invention
This invention relates to methods and compositions for the treatment of insulin resistance.
2. Description of Related Art
Insulin resistance is associated with several disease conditions including non-insulin dependent diabetes mellitus (NIDDM), obesity, hypertension, and cardiovascular disease. The most well-studies of these conditions is NIDDM. NIDDM, also termed maturity-onset diabetes or type II diabetes to differentiate it from insulin-dependent diabetes mellitus (IDDM, also termed type I or juvenile diabetes), usually occurs in middle-aged obese people and accounts for 80% to 90% of diagnosed diabetes. In addition to insulin resistance, NIDDM is associated with normal to elevated levels of insulin, hyperglycemia, increased levels of very low density lipoproteins (VLDL), and decreased muscle uptake of glucose. NIDDM is often associated with hypertriglyceridemia. Ketoacidosis, characteristic of IDDM, is not associated with NIDDM except when the patient is subjected to extreme stress (e.g., septic shock or myocardial infarction). NIDDM patients tend to develop many of the same complications associated with IDDM including nerve, eye, kidney, and coronary artery disease.
Mounting scientific evidence suggests that NIDDM results from a combination of two components: 1) a hereditary, genetic component (Rotter et al. In:: Rifkin et al., Diabetes Mellitus: Theory and Practice., New York, Elsevier, 1990, pp. 378-413); and 2) an acquired component (Seely et al. In: Moller, Ed. Insulin Resistance and Its Clinical Disorders. England, John Wile and Sons, Ltd., 1993, pp. 187-252; Olefsky In: Efendic, et al. Eds. New Concepts in the Pathogenesis of NIDDM. New York, Plenum Publishing Corp., 1993; Olefsky, In: DeGroot, et al., Eds. DeGroot Textbook of Endocrinology., 3rd Ed., Philadelphia, W. B. Saunders and Co., 1994). The genetic component of NIDDM is responsible for the first stage of the disease, termed the xe2x80x9cprediabeticxe2x80x9d state. The prediabetic state is characterized by hyperinsulinemia and xe2x80x9cprimaryxe2x80x9d insulin resistance. Insulin responsiveness in the prediabetic state is sufficient to maintain normal glucose tolerance (NGT) or at least impaired glucose tolerance (IGT).
As time passes, this compensatory mechanism fails in a subset of subjects, due to a decline in function of the insulin-producing xcex2 cells of the pancreas. The decline in insulin secretion, superimposed on the pre-existing genetic background, leads to the development of hyperglycemia, increased xe2x80x9csecondaryxe2x80x9d insulin-resistance, and the final diabetic NIDDM state. The increase in insulin resistance in diabetic NIDDM relative to prediabetic NIDDM suggests that an additional, non-inherited factor creates a secondary component of insulin resistance that is additive to the inherited insulin resistance-inducing component present in the prediabetic state. This additional factor is hyperglycemia (Yki-Jarvinen, 1990, Diabetologia, 33:579-585; Yki-Jarvinen, 1992, Endocrine Rev., 13:415-431).
The precise mechanism through which hyperglycemia induces insulin resistance is not understood. However, several observations have suggested that insulin resistance is due, at least in part, due to inhibition of the normal insulin receptor function. First, the hyperglycemic-NIDDM state leads to reduced insulin-stimulated activities including insulin receptor autophosphorylation, insulin receptor-mediated kinase activities (including tyrosine kinase), insulin-stimulated phosphatidylinositol kinase activity, and insulin-stimulated DNA synthesis (Freidenberg et al., 1987, J. Clin. Invest., 79:240-250; Caro et al., 1986, J. Clin. Invest., 78:249-258; Comi et al., 1987, J. Clin. Invest., 79:453-462; Caro et al., 1987, J. Clin. Invest., 79:1330-1337). Thus, although the receptor can bind insulin, the normal insulin-mediated transduction signals are not transmitted. The decrease in insulin receptor kinase activity has been correlated with the magnitude of the patient""s hyperglycemia (Nolan et al., 1994, J. Clin. Endocrinol. Metab., 78:471-477; Brillon et al., 1989, Diabetes, 38:397-403; Maegawa et al., 1991, Diabetes, 40:815-819).
Secondly, protein kinase C (PKC), which can phosphorylate serine and/or threonine residues, has been implicated in inactivation of insulin receptors in vitro. Incubation of cells under hyperglycemic conditions induces translocation and activation of PKC (Mxc3xcller et al., 1991, Diabetes, 40:1440-1448; Mosthaf et al., 1993, Exp. Clin. Endocrinol., 101(Suppl 2):150-151). Phorbol ester-mediated induction of PKC serine phosphorylation activity decreases insulin receptor kinase activity (Takayama et al., 1988, J. Biol. Chem., 263:3440-3447). Hyperglycemia-induced inhibition of insulin receptor kinase activity is inhibited by incubation of cells with broad-based, non-specific PKC inhibitors such as staurosporin, H7, and polymyxin B (Mosthaf et al., supra).
The specific site of PKC-mediated phosphorylation is unknown. A better understanding of the cellular mechanisms underlying hyperglycemia-induced insulin resistance would greatly facilitate the design and development of specific therapeutics.
The invention is based on the discovery that hyperglycemia causes protein kinase C (PKC) to aberrantly phosphorylate a specific serine residue (Ser1270) of insulin receptors. While phosphorylation of insulin receptor Ser1270 does not significantly affect insulin binding, phosphorylation of Ser1270 inhibits the insulin receptor""s autophosphorylation and tyrosine kinase activities, thus inhibiting transduction of insulin-stimulated intracellular signals. The inability of the serine-phosphorylated insulin receptor to respond to insulin binding can result in insulin resistance.
In general, the invention features compositions, and methods for their identification and use in the inhibition of phosphorylation of insulin receptor residue Ser1270 by protein kinase C (PKC).
In one aspect the invention features a protein kinase C antagonist having activity in inhibition of protein kinase C-mediated phosphorylation of Ser1270 of an insulin receptor.
The invention also features a method of testing a candidate compound for PKC antagonist activity. According to the method, the candidate compound is contacted with i) protein kinase C and ii) a substrate for protein kinase C. A preferred substrate is a polypeptide derived from an insulin receptor, and containing amino acid sequences flanking Ser1270. Compounds having PKC antagonist activity are identified by detecting a level of polypeptide substrate phosphorylation in the presence of the candidate compound.
In related aspects, the invention features a purified polynucleotide encoding a polypeptide having the amino acid sequence DDLHPSFPEVS (SEQ ID NO:1), with the proviso that polynucleotide does not encode a native insulin receptor, as well as vectors and transformed host cells containing the polynucleotide.
The invention additionally features therapeutic compositions composed of a PKC antagonist of the invention and a pharmaceutically acceptable carrier.
The therapeutic compositions of the invention can be used in a method of treating insulin resistance in a patient by administering a therapeutic composition containing a PKC antagonist in an amount effective to inhibit PKC-mediated serine phosphorylation of insulin receptors of the patient.
The invention further features a polynucleotide encoding a serine phosphorylation-resistant human insulin receptor, as well as vectors and host cells containing the polynucleotide.
The polynucleotide encoding a serine phosphorylation-resistant human insulin receptor can be used in a method of treatment of an insulin-resistant patient by genetically transforming cells of a patient with a construct containing the nucleotide sequence encoding a serine phosphorylation-resistant human insulin receptor, and a eukaryotic promoter sequence operably linked to the nucleotide sequence.
One advantage of the invention is that the PKC antagonists of the invention need only be delivered to insulin receptor-expressing cells in order to restore insulin sensitivity in the patient. Restoration of patient insulin sensitivity does not require complete inhibition of PKC-mediated insulin receptor serine phosphorylation, or even restoration of insulin receptor responsiveness in all insulin receptor-expressing cells.
Another advantage of the invention is that the polypeptide substrate of PKC serine phosphorylation (i.e., the insulin receptor polypeptide containing Ser1270) provides a rationale basis for the design of drugs for the treatment of insulin resistance via the inhibition of PKC-mediated insulin receptor serine phosphorylation.
Still another advantage of the invention is that inhibition of PKC-mediated Ser1270 phosphorylation serves as the basis for a rapid screening assay to identify candidate compounds which have activity as PKC serine phosphorylation inhibitors.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.